The existence of the apoptotic pathway can be both vital and devastating for organisms. While regulated cell death by apoptosis is essential during development and for maintaining homeostasis in organisms, dysregulation of apoptosis is associated with numerous pathological conditions including cancer, neurodegeneration and cardiovascular diseases. Therefore, a cell's ability to suppress the apoptotic pathway, activating it only when necessary, is indeed a delicate balance. Our hypothesis is that there are fundamental differences in how the apoptotic pathway is regulated in mitotic versus postmitotic cells. We propose that in postmitotic cells such as neurons, cardiomyocytes, and myotubes, which have limited regenerative potential and last for the lifetime of organisms, apoptosis is regulated more strictly than in mitotic cells. We have focused our studies on the regulation of cytochrome c- dependent caspase activation because this event is a crucial point of no return for cell death in mammalian cells. We find that whereas cytosolic cytochrome c alone is sufficient to induce apoptosis in many mitotic cells, it is not capable of doing so in the postmitotic neurons, cardiomyocytes and myotubes because of strict control of caspase activation by XIAP. We have also identified another unexpected checkpoint where we find levels of endogenous cytochrome c to be limiting for apoptosis in postmitotic but not mitotic cells. In Specific Aim 1, we will investigate how endogenous XIAP is able to selectively regulate apoptosis in postmitotic but not mitotic cells. We will also examine how XIAP's strict inhibition of caspases is relieved to permit apoptosis in postmitotic cells. In Specific Aim 2, we will examine the mechanism by which endogenous levels of cytochrome c become rate-limiting for apoptosis in postmitotic but not mitotic cells. We will also test the hypothesis that apoptotic signals in postmitotic cells elevate endogenous cytochrome c levels above the threshold necessary for activating caspases. In Specific Aim 3, we focus our attention on senescent cells. We will examine whether the development of senescence engages changes in the regulation of cytochrome c-dependent apoptosis as seen in the terminally-differentiated cells. Understanding how apoptosis is differentially regulated in mitotic and postmitotic cells is clinically significant because it identifies drug targets that could inhibit or activate apoptosis in selective cell types.